The TNF superfamily death ligand TRAIL is emerging as a potential tool in the treatment of many cancers based on its ability to selectively kill cancer cells and repress tumor metastasis. Unfortunately, many tumor cell lines are resistant to TRAIL, suggesting the molecular basis of TRAIL action and how TRAIL-sensitivity can be restored is crucial for maximizing the potential of this promising cancer therapeutic. The long-term goal is to understand precisely how cancer cells become resistant to TRAIL therapy and how this knowledge can, in turn, lead to novel therapeutic interventions that restore TRAIL-sensitivity. The objective in this particular application is to determine how TRAIL kills cancer cells by switching the multifunctional sorting protein PACS-2 to an apoptotic effector and how dysregulation of PACS-2 enables cancers to resist TRAIL-induced cell death. Specifically, Akt-phosphorylated PACS-2 mediates homeostasis in healthy cells by coordinating the localization of antiapoptotic calcium channels to the endoplasmic reticulum (ER) with ER-mitochondria communication. In response to TRAIL, PACS-2 becomes dephosphorylated, promoting mitochondria permeabilization and cell death by coordinating apoptotic calcium signaling with lysosome-mitochondria communication that mediates Bid activation. The central hypothesis is that dysregulation of PACS-2 enables cancer cells to resist TRAIL killing in two different ways: in cancers with elevated Akt, PACS-2 apoptotic activity is repressed by persistent Akt phosphorylation, whereas in cancers with loss of the PACS-2 locus, TRAIL-induced apoptosis is repressed independent of Akt status. The rationale for the proposed research is that successful completion will establish a causal relationship between PACS-2 dysregulation and tumor progression and will determine how TRAIL switches PACS-2 to an apoptotic mediator that coordinates the complex interorganellar communication required for mitochondria permeabilization and cancer cell death. Guided by strong preliminary data, this hypothesis will be test- ed by pursuing three specific aims: 1) Determine to what extent loss or repression of PACS-2 accelerates tumor progression, 2) Identify the mechanism used by TRAIL to induce apoptotic activation of PACS-2 to mediate ER-mitochondria calcium signaling, and 3) Determine how TRAIL induces PACS-2 to coordinate lysosome membrane permeabilization with cathepsin B-mediated Bid cleavage on mitochondria. The approach is innovative because it uses a comprehensive, multi-disciplinary design to dissect the molecular basis of TRAIL action. The proposed research is significant because it presents a novel and testable model of how PACS-2 acts as a molecular switch to integrate cell homeostasis with TRAIL-induced apoptosis and, therefore, how PACS-2 dysregulation may accelerate tumor progression and cause resistance to TRAIL therapy.